The proposed research is a continuation of our long-range program directed towards the development of effective and practical procedures and techniques needed for banking granulocytes for clinical use. Our new technique of collecting blood without the use of anticoagulants, combined with our centrifugal elutriation method for isolation of pure granulocytes, offers a unique source of granulocytes in an optimal physiological condition. Using these cells, we propose to investigate several new approaches to short-term and long-term banking of granulocytes. We will test the following hypotheses: (1) The existence of a correlation between lysosomal damage (as measured by the release of beta-glucuronidase) and loss of cell function after preservation. Agents known to stabilize lysosomal membranes or inhibit the activity of their degradative enzymes will be screened for their effectiveness in reducing cell damage; (2) The possibility of increasing the stability of the granulocyte cell membrane by modifying its lipid content by means of incubation with liposomes; (3) The invlvement of -SH groups in granulocyte damage during preservation. Agents known to prevent -SH oxidation will be screened for their effectiveness in preventing or repairing such damage if it occurs. Also, the nature of the factor(s) in serum which increase(s) granulocyte susceptability to freezing damage will be studied. The preservation techniques to be evaluated include liquid storage of granulocytes both above and below 0 degrees C (the latter being under elevated hydrostatic pressure) and cryopreservation either by conventional slow freezing or by a two-step rapid freezing procedure. The functionability of preserved granulocytes will be assayed both in vitro, by testing their membrane integrity, phagocytic acitivity and bactericidal function, and in vivo, by the skinwindow technique. The latter will be done in cooperation with Drs. William H. Stone and James Congdon at Letterman Army Hospital. In addition, the development of several new techniques for in vitro assessment of granulocytes function is proposed.